Kinase-mediated regulation of common transcription factors accounts for the bone-protective effects of sex steroids

Abstract

IntroductionEstrogens and androgens exert regulatory influences on a wide variety of biological processes by signaling through highly specialized proteins belonging to the superfamily of nuclear receptors: the estrogen receptors (ERs) α and β and the androgen receptor (AR) (1-3). Binding of the hormone to the receptor causes homo-or heterodimerization of the protein and changes its conformation in such a way that it allows the dimer to interact with several coactivator proteins. The receptor-coactivator(s) complex attaches to specific DNA response elements (estrogen response elements [EREs] and androgen response elements [AREs]) and the basal transcription machinery, causing histone acetylation, decondensation of the chromatin, and initiation of transcription. Estrogens and androgens also regulate the transcription of genes that do not contain EREs or AREs. In this case the ligandactivated receptors form protein-protein complexes with other transcription factors, thus preventing them from interacting with their target gene promoters (4).Nonetheless, numerous effects of estrogens and androgens cannot be explained by the established models of transcriptional regulation resulting from cis-or trans-interactions of their classical nuclear receptor proteins with DNA. To distinguish them from the classical, It has been found that 4-estren-3α,17β-diol, a synthetic ligand for the estrogen receptor (ER) or androgen receptor (AR), which does not affect classical transcription, reverses bone loss in ovariectomized females or orchidectomized males without affecting the uterus or seminal vesicles, demonstrating that the classical genotropic actions of sex steroid receptors are dispensable for their bone-protective effects, but indispensable for their effects on reproductive organs. We have now investigated the mechanism of action of this compound. We report that, identically to 17β-estradiol or dihydrotestosterone, but differently from raloxifene, estren alters the activity of Elk-1, CCAAT enhancer binding protein-β (C/EBPβ), and cyclic adenosine monophosphate-response element binding protein (CREB), or c-Jun/ c-Fos by an extranuclear action of the ER or AR, resulting in activation of the Src/Shc/ERK pathway or downregulation of JNK, respectively. All of these effects are non-sex specific, require only the ligandbinding domain of the receptor, and are indispensable for the antiapoptotic action of these ligands on osteoblastic and HeLa cells. Moreover, administration of 17β-estradiol or 4-estren-3α,17β-diol to ovariectomized mice induces phosphorylation of ERKs, Elk-1, and C/EBPβ, downregulates c-Jun, and upregulates the expression of egr-1, an ERK/SRE target gene. Kinase-initiated regulation of commonly used transcription factors offers a molecular explanation for the profound skeletal effects of sex steroid receptor ligands, including synthetic ones that are devoid of classical transcriptional activity.

Estrogen receptors outside the nucleus in breast cancer

“…When the E domain of ERa or the full-length receptor is targeted to the nucleus, no rapid activation of ERK is detected [9]. Instead, when either construct is targeted exclusively to the plasma membrane, multiple rapid signals are generated in response to E2 [61,70]. Thus, the available evidence suggests that membrane localization of ER is required for kinase activation.…”

Genomic and non-genomic actions of sex steroids in the growth plate

“…In contrast, E 2 administration by a subcutaneously implanted slow-release pellet caused a strong increase in trabecular bone volume in both females and males. Remarkably, the non-genomic component DE had no effect on bone mass accrual, suggesting that non-genomic signaling is not involved in this process, in marked contrast to its proposed protective role in the remodeling of the skeleton during postmenopausal osteoporosis [46,49,50,51,52]. These data suggest that before sexual maturation ERs may play a significant role in the regulation of bone mass accrual, although the source of the ligands is largely unclear.…”

“…They can operate as ligand-induced transcription factors in the so-called genomic signaling cascade or they can work as activators of non-genomic signaling. In this way, E 2 activates putative membrane-bound ERs, which activate a downstream signaling cascade involving kinases such as Src, Shc, and Erk [49,50,51]. This latter pathway has been proposed to operate in osteoblasts and osteocytes and to be responsible for the protective effect of E 2 on postmenopausal bone loss by preventing osteoblast and osteocyte apoptosis [51,52].…”

“…In this way, E 2 activates putative membrane-bound ERs, which activate a downstream signaling cascade involving kinases such as Src, Shc, and Erk [49,50,51]. This latter pathway has been proposed to operate in osteoblasts and osteocytes and to be responsible for the protective effect of E 2 on postmenopausal bone loss by preventing osteoblast and osteocyte apoptosis [51,52]. In cultured chondrocytes it has also been suggested that membrane-bound ERs might be present and influence chondrocyte activity in a nongenomic manner [53].…”

“…In analogy to the experiments in the growth plate, we assessed whether receptors and steroidogenic enzymes were expressed in rat metaphyseal bone at representative stages after birth, including before, during, and after sexual maturation, to better define the role of E 2 in modulating bone mass accrual. Several studies have demonstrated ERs in bone cells [34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55]. However, reports on the expression of steroidogenic enzymes in bone cells are limited to in vitro studies in osteoblast-like cells and primary osteoblasts [28,29,56].…”